Human immune interferon

ABSTRACT

Disclosed is a complete description of the preparation of novel, substantially pure polypeptide via recombinant DNA techniques utilizing any of an assortment of expression vectors and host cultures. The polypeptide, human immune (gamma) interferon (IFN-γ), is isolated and characterized in terms of DNA and amino acid sequences, physical attributes and biological activity.

This is a division of application Ser. No. 746,813, filed June 20, 1985,pending, which is a continuation of application Ser. No. 312,489, filedOct. 19, 1981, now abandoned.

FIELD OF THE INVENTION

The present invention relates to the field of recombinant DNAtechnology, to means and methods utilizing such technology in thediscovery of the DNA sequence and deduced amino acid sequence for humanimmune interferon and to its production and to the various products ofsuch production and their uses.

More particularly, the present invention relates to the isolation andidentification of DNA sequences encoding human immune interferon and tothe construction of recombinant DNA expression vehicles containing suchDNA sequences operably linked to expression-effecting promoter sequencesand to the expression vehicles so constructed. In another aspect, thepresent invention relates to host culture systems, such as variousmicroorganism and vertebrate cell cultures transformed with suchexpression vehicles and thus directed in the expression of the DNAsequences referred to above. In yet other aspects, this inventionrelates to the means and methods of converting the end products of suchexpression to novel entities, such as pharmaceutical compositions,useful for the prophylactic or therapeutic treatment of humans. Inpreferred embodiments, this invention provides particular expressionvehicles that are sequenced properly such that human immune interferonis produced and secreted from the host cell in mature form. In addition,this invention relates to various processes useful for producing saidDNA sequences, expression vehicles, host culture systems and endproducts and entities thereof and to specific and associated embodimentsthereof.

The present invention arises in part from the discovery of the DNAsequence and deduced amino acid sequence encoding human immuneinterferon. In addition, the present invention provides sequenceinformation on the 3'- and 5'-flanking sequences of the human immuneinterferon gene, facilitating the in vitro linkage thereof intoexpression vehicles. In particular, there is provided the 5'-DNA segmentencoding the putative endogenous signal polypeptide which immediatelyprecedes the amino acid sequence of the putative mature human immuneinterferon. These discoveries, in turn, have enabled the development ofthe means and methods for producing, via recombinant DNA technology,sufficient amounts of human immune interferon, so as to enable, in turn,the determination of its biochemical properties and bioactivity. Thepublications and other materials hereof used to illuminate thebackground of the invention, and in particular cases, to provideadditional details respecting its practice are incorporated herein byreference, and for convenience, are numerically referenced by thefollowing text and respectively grouped in the appended bibliography.

BACKGROUND OF THE INVENTION A. Human Immune Interferon

Human interferons can be classified in three groups on the basis ofdifferent antigenicity and biological and biochemical properties.

The first group comprises a family of leukocyte interferons(α-interferon, LeIF or IFN-α), which are normally produced mainly byconstituent cells of human blood upon viral induction. These have beenmicrobially produced and found to be biologically active (1, 2, 3).Their biological properties have prompted their use in the clinic astherapeutic agents for the treatment of viral infections and malignantconditions (4).

In the second group is human fibroblast interferon (β-interferon, FIF orIFN-β), normally produced by fibroblasts upon viral induction, which haslikewise been microbially produced and found to exhibit a wide range ofbiological activities (5). Clinical trials also indicate its potentialtherapeutic value. The leukocyte and fibroblast interferons exhibit veryclear similarities in their biological properties despite the fact thatthe degree of homology at the amino acid level is relatively low. Inaddition, both groups of interferons contain from 165 to 166 amino acidsand are acid stable proteins.

The human immune interferon (γ-interferon, IIF or IFN-γ), to which thisinvention is directed, is, in contrast to the α- and β-interferons, pH 2labile, is produced mainly upon mitogenic induction of lymphocytes andis also clearly antigenically distinct. Until recently human immuneinterferon could only be detected in very minor levels, which evidentlyhampered its characterization. Recently, a rather extensive but stillpartial purification of human immune interferon has been reported (6).The compound was said to be produced from lymphocyte cultures stimulatedwith a combination of phytohaemagglutin and a phorbol ester and purifiedby sequential chromatographic separations. This procedure resulted in aproduct having a molecular weight of 58,000.

Human immune interferon has been produced in very low amounts bytranslating mRNA in oocytes, showing interferon activity characteristicof human immune interferon and expressing the hope that immuneinterferon cDNA could be synthesized and cloned (7).

The amount of immune interferon obtained until now is certainlyinsufficient to carry out unambiguous experiments on thecharacterization and biological properties of the purified component.However, in vitro studies performed with crude preparations, as well asin vivo experiments with murine γ-interferon preparations, suggest thatthe primary function of immune interferon may be as an immunoregulatoryagent (8, 9). Immune interferon has not only an antiviral andanticellular activity in common to all human interferons, but shows apotentiating effect on these activities with α- and β-interferon (10).Also, the in vitro antiproliferative effect of γ-interferon on tumorcells is reported to be approximately 10- to 100-fold that of the otherinterferon classes (8, 11, 12). This result, together with itspronounced immunoregulatory role (8, 9), suggests a much more pronouncedantitumoral potency for IFN-γ than for IFN-α and IFN-β. Indeed, in vivoexperiments with mice and murine IFN-γ preparations show a clearsuperiority over antivirally induced interferons in its antitumoraleffect against osteogenic sarcoma (13).

All of these studies, until the present invention, had to be performedwith rather crude preparations, due to the very low availability.However, they certainly suggest very important biological functions forimmune interferon. Not only has immune interferon a potent associatedantiviral activity, but probably also a strong immunoregulatory andantitumoral activity, clearly pointing to a potentially very promisingclinical candidate.

It was perceived that the application of recombinant DNA technologywould be a most effective way of providing the requisite largerquantities of human immune interferon. Whether or not the materials soproduced would include glycosylation which is considered characteristicof native, human derived material, they would probably exhibitbioactivity admitting of their use clinically in the treatment of a widerange of viral, neoplastic, and immunosuppressed conditions or diseases.

B. Recombinant DNA Technology

Recombinant DNA technology has reached the age of some sophistication.Molecular biologists are able to recombine various DNA sequences withsome facility, creating new DNA entities capable of producing copiousamounts of exogenous protein product in transformed microbes. Thegeneral means and methods are in hand for the in vitro ligation ofvarious blunt ended or "sticky" ended fragments of DNA, producing potentexpression vehicles useful in transforming particular organisms, thusdirecting their efficient synthesis of desired exogenous product.However, on an individual product basis, the pathway remains somewhattortuous and the science has not advanced to a stage where regularpredictions of success can be made. Indeed, those who portend successfulresults without the underlying experimental basis, do so withconsiderable risk of inoperability.

The plasmid, a nonchromosomal loop of double-stranded DNA found inbacteria and other microbes, oftentimes in multiple copies per cell,remains a basic element of recombinant DNA technology. Included in theinformation encoded in the plasmid DNA is that required to reproduce theplasmid in daughter cells (i.e., an origin of replication) andordinarily, one or more phenotypic selection characteristics such as, inthe case of bacteria, resistance to antibiotics, which permit clones ofthe host cell containing the plasmid of interest to be recognized andpreferentially grown in selective media. The utility of plasmids lies inthe fact that they can be specifically cleaved by one or anotherrestriction endonuclease or "restriction enzyme", each of whichrecognizes a different site on the plasmid DNA. Thereafter heterologousgenes or gene fragments may be inserted into the plasmid by endwisejoining at the cleavage site or at reconstructed ends adjacent to thecleavage site. Thus formed are so-called replicable expression vehicles.DNA recombination is performed outside the cell, but the resulting"recombinant" replicable expression vehicle, or plasmid, can beintroduced into cells by a process known as transformation and largequantities of the recombinant vehicle obtained by growing thetransformant. Moreover, where the gene is properly inserted withreference to portions of the plasmid which govern the transcription andtranslation of the encoded DNA message, the resulting expression vehiclecan be used to actually produce the polypeptide sequence for which theinserted gene codes, a process referred to as expression.

Expression is initiated in a region known as the promoter which isrecognized by and bound by RNA polymerase. In the transcription phase ofexpression, the DNA unwinds, exposing it as a template for initiatedsynthesis of messenger RNA from the DNA sequence. The messenger RNA is,in turn, translated into a polypeptide having the amino acid sequenceencoded by the mRNA. Each amino acid is encoded by a nucleotide tripletor "codon" which collectively make up the "structural gene", i.e. thatpart which encodes the amino acid sequence of the expressed polypeptideproduct. Translation is initiated at a "start" signal (ordinarily ATG,which in the resulting messenger RNA becomes AUG). So-called stop codonsdefine the end of translation and, hence, of production of further aminoacid units. The resulting product may be obtained by lysing, ifnecessary, the host cell, in microbial systems, and recovering theproduct by appropriate purification from other proteins.

In practice, the use of recombinant DNA technology can express entirelyheterologous polypeptides--so-called direct expression--or alternativelymay express a heterologous polypeptide fused to a portion of the aminoacid sequence of a homologous polypeptide. In the latter cases, theintended bioactive product is sometimes rendered bioinactive within thefused, homologous/heterologous polypeptide until it is cleaved in anextracellular environment. See British Pat. Publ. No. 2007676A andWetzel, American Scientist 68, 664 (1980).

C. Cell Culture Technology

The art of cell or tissue cultures for studying genetics and cellphysiology is well established. Means and methods are in hand formaintaining permanent cell lines, prepared by successive serialtransfers from isolate normal cells. For use in research, such celllines are maintained on a solid support in liquid medium, or by growthin suspension containing support nutriments. Scale-up for largepreparations seems to pose only mechanical problems. For furtherbackground, attention is directed to Microbiology, 2nd Edition, Harperand Row, Publishers, Inc, Hagerstown, Md. (1973) especially pp. 1122 etseq. and Scientific American 245, 66 et seq. (1981), each of which isincorporated herein by this reference.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that recombinant DNAtechnology can be used to successfully produce human immune interferon,preferably in direct form, and in amounts sufficient to initiate andconduct animal and clinical testing as prerequisites to market approval.The product is suitable for use, in all of its forms, in theprophylactic or therapeutic treatment of human beings for viralinfections and malignant and immunosuppressed or immunodeficientconditions. Its forms include various possible oligomeric forms whichmay include associated glycosylation. The product is produced bygenetically engineered transformant microorganisms or transformant cellculture systems. As used herein, the term "transformant cell" refers toa cell into which has been introduced DNA, said DNA arising fromexogenous DNA recombination, and to the progeny of any such cell whichretains the DNA so introduced. Thus, the potential now exists to prepareand isolate human immune interferon in a more efficient manner than hasbeen possible. One significant factor of the present invention, in itsmost preferred embodiments, is the accomplishment of geneticallydirecting a microorganism or cell culture to produce human immuneinterferon in isolatable amounts, secreted from the host cell in matureform.

The present invention comprises the human immune interferon thusproduced and the means and methods of its production. The presentinvention is further directed to replicable DNA expression vehiclesharboring gene sequences encoding human immune interferon in expressibleform. Further, the present invention is directed to microorganismstrains or cell cultures transformed with the expression vehiclesdescribed above and to microbial or cell cultures of such transformedstrains or cultures, capable of producing human immune interferon. Instill further aspects, the present invention is directed to variousprocesses useful for preparing said immune interferon gene sequences,DNA expression vehicles, microorganism strains and cell cultures and tospecific embodiments thereof. Still further, this invention is directedto the preparation of fermentation cultures of said microorganisms andcell cultures. In addition, this invention is directed to thepreparation of human immune interferon, as a direct expression product,secreted from the host cell in mature form. This approach may utilizethe gene encoding the sequence of the mature human immune interferonplus the 5' flanking DNA encoding the signal polypeptide. The signalpolypeptide is believed to aid in the transport of the molecule to thecellular wall of the host organisms where it is cleaved during thesecretion process of the mature human interferon product. Thisembodiment enables the isolation and purification of the intended matureimmune interferon without resort to involved procedures designed toeliminate contaminants of intracellular host protein or cellular debris.

Reference herein to the expression "mature human immune interferon"connotes the microbial or cell culture production of human immuneinterferon unaccompanied by the signal peptide or presequence peptidethat immediately attends translation of the human immune interferonmRNA. A first recombinant human immune interferon, according to thepresent invention, is thus provided, having methionine as its firstamino acid (present by virtue of the ATG start signal codon insertion infront of the structural gene) or, where the methionine is intra- orextracellularly cleaved, having its normally first amino acid cysteine.Mature human immune interferon can also be produced, in accordanceherewith, together with a conjugated protein other than the conventionalsignal polypeptide, the conjugate being specifically cleavable in anintra- or extracellular environment. See British Pat. publication No.2007676A. Finally, the mature human immune interferon can be produced bydirect expression without the necessity of cleaving away any extraneous,superfluous polypeptide. This is particularly important where a givenhost may not, or not efficiently, remove a signal peptide where theexpression vehicle is designed to express the mature human interferontogether with its signal peptide. The thus produced mature human immuneinterferon is recovered and purified to a level fitting it for use inthe treatment of viral, malignant, and immunosuppressed orimmunodeficient conditions.

Human immune interferon was obtained according to the following:

1. Human tissues, for example human spleen tissue or peripheral bloodlymphocytes, were cultured with mitogens to stimulate the production ofimmune interferon.

2. Cell pellets from such cell cultures were extracted in the presenceof ribonuclease inhibitor to isolate all cytoplasmic RNA.

3. An oligo-dT column isolated the total messenger RNA (mRNA) inpolyadenylated form. This mRNA was size-fractionated using sucrosedensity gradient and acid-urea gel electrophoresis.

4. The appropriate mRNA (12 to 18 S) was converted to correspondingsingle stranded complementary DNA (cDNA) from which was produced doublestranded cDNA. After poly-dC tailing, it was inserted into a vector,such as a plasmid bearing one or more phenotypic markers.

5. The thus prepared vectors were used to transform bacterial cellsproviding a colony library. Radiolabeled cDNA prepared from both inducedand uninduced mRNA, derived as described above, was used to separatelyprobe duplicate colony libraries. The excess cDNA was then removed andthe colonies exposed to X-ray film so as to identify the induced cDNAclones.

6. From the induced cDNA clones the corresponding plasmid DNA wasisolated and sequenced.

7. In a first embodiment sequenced DNA was then tailored in vitro forinsertion into an appropriate expression vehicle which was used totransform an E. coli host cell which was, in turn, permitted to grow ina culture and to express the desired human immune interferon product.

8. Human immune interferon thus expressed doubtless has 146 amino acidsin its mature form, beginning with cysteine, and is very basic incharacter. Its monomeric molecular weight has been calculated at 17,140.Perhaps because of the presence of numerous basic residues,hydrophobicity, salt bridge formation and so forth, the molecule mayassociate itself in oligomeric forms, e.g., in dimer, trimer or tetramerform. The high molecular weights previously observed with naturalmaterial (6) which can not be accounted for on the basis of the aminoacid sequence alone may be due to such oligomeric forms as well as tothe contribution of carbohydrate from post-translational glycosylation.

9. In certain host cell systems, particularly when ligated into anexpression vehicle so as to be expressed together with its signalpeptide, the mature form of human immune interferon is exported into thecell culture medium, immeasurably aiding in recovery and purificationmethods.

DESCRIPTION OF PREFERRED EMBODIMENTS A. Microorganisms/Cell Cultures

1. Bacterials Strains/Promoters

The work described herein was performed employing, inter alia, themicroorganism E. coli K-12 strain 294 (end A, thi⁻, hsr⁻, _(k) hsm⁺), asdescribed in British Pat. Publication No. 2055382 A. This strain hasbeen deposited with the American Type Culture Collection, ATCC AccessionNo. 31446. However, various other microbial strains are useful,including known E. coli strains such as E. coli B, E. coli X 1776 (ATCCNo. 31537) and E. coli W 3110 (F⁻, λ⁻, protrophic) (ATCC No. 27325), orother microbial strains many of which are deposited and (potentially)available from recognized microorganism depository institutions, such asthe American Type Culture Collection (ATCC)--cf. the ATCC cataloguelisting. See also German Offenlegungsschrift No. 2644432. These othermicroorganisms include, for example, Bacilli such as Bacillus subtilisand other enterobacteriaceae among which can be mentioned as examplesSalmonella typhimurium and Serratia marcesans, utilizing plasmids thatcan replicate and express heterologous gene sequences therein.

As examples, the beta lactamase and lactose promoter systems have beenadvantageously used to initiate and sustain microbial production ofheterologous polypeptides. Details relating to the make-up andconstruction of these promoter systems have been published by Chang etal., Nature 275, 617 (1978) and Itakura et al., Science 198, 1056(1977), which are hereby incorporated by reference. More recently, asystem based upon tryptophan, the so-called trp promoter system, hasbeen developed. Details relating to the make-up and construction of thissystem have been published by Goeddel et al., Nucleic Acids Research 8,4057 (1980) and Kleid et al., U.S. Ser. No. 133, 296, filed Mar. 24,1980, which are hereby incorporated by reference. Numerous othermicrobial promoters have been discovered and utilized and detailsconcerning their nucleotide sequences, enabling a skilled worker toligate them functionally within plasmid vectors, have beenpublished--see, e.g., Siebenlist et al., Cell 20, 269 (1980), which isincorporated herein by this reference.

2. Yeast Strains/Yeast Promoters

The expression system hereof may also employ the plasmid YRp7 (14, 15,16), which is capable of selection and replication in both E. coli andthe yeast, Saccharomyces cerevisiae. For selection in yeast the plasmidcontains the TRP1 gene (14, 15, 16) which complements (allows for growthin the absence of tryptophan) yeast containing mutations in this genefound on chromosome IV of yeast (17). The strain used here was thestrain RH218 (18) deposited at the American Type Culture Collectionwithout restriction (ATCC No. 44076). However, it will be understoodthat any Saccharomyces cerevisiae strain containing a mutation whichmakes the cell trp1 should be an effective environment for expression ofthe plasmid containing the expression system. An example of anotherstrain which could be used is pep4-1 (19). This tryptophan auxotrophstrain also has a point mutation in TRP1 gene.

When placed on the 5' side of a non-yeast gene the 5'-flanking DNAsequence (promoter) from a yeast gene (for alcohol dehydrogenase 1) canpromote the expression of a foreign gene in yeast when placed in aplasmid used to transform yeast. Besides a promoter, proper expressionof a non-yeast gene in yeast requires a second yeast sequence placed atthe 3'-end of the non-yeast gene on the plasmid so as to allow forproper transcription termination and polyadenylation in yeast. Thispromoter can be suitably employed in the present invention as well asothers--see infra. In the preferred embodiments, the 5'-flankingsequence of the yeast 3-phosphoglycerate kinase gene (20) is placedupstream from the structural gene followed again by DNA containingtermination--polyadenylation signals, for example, the TRP1 (14, 15, 16)gene or the PGK (20) gene.

Because yeast 5'-flanking sequence (in conjunction with 3' yeasttermination DNA) (infra) can function to promote expression of foreigngenes in yeast, it seems likely that the 5'-flanking sequence of anyhighly-expressed yeast gene could be used for the expression ofimportant gene products. Since under some circumstances yeast expressedup to 65 percent of its soluble protein as glycolytic enzymes (21) andsince this high level appears to result from the production of highlevels of the individual mRNAs (22), it should be possible to use the5'-flanking sequences of any other glycolytic genes for such expressionpurposes--e.g., enolase, glyceraldehyde--3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose--6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Any of the 3'-flanking sequences of these genes could alsobe used for proper termination and mRNA polyadenylation in such anexpression system--cf. Supra. Some other highly expressed genes arethose for the acid phosphatases (23) and those that express high levelsof production due to mutations in the 5'-flanking regions (mutants thatincrease expression)--usually due to the presence of a TYl transposableelement (24).

All of the genes mentioned above are thought to be transcribed by yeastRNA polymerase II (24). It is possible that the promoters for RNApolymerase I and III which transcribe genes for ribosomal RNA, 5S RNA,and tRNAs (24, 25), may also be useful in such expression constructions.

Finally, many yeast promoters also contain transcriptional control sothey may be turned off or on by variation in growth conditions. Someexamples of such yeast promoters are the genes that produce thefollowing proteins: Alcohol dehydrogenase II, isocytochrome-c, acidphosphatase, degradative enzymes associated with nitrogen metabolism,glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization (22). Such a control region would bevery useful in controlling expression of protein product--especiallywhen their production is toxic to yeast. It should also be possible toput the control region of one 5'-flanking sequence with a 5'-flankingsequence containing a promoter from a highly expressed gene. This wouldresult in a hybrid promoter and should be possible since the controlregion and the promoter appear to be physically distinct DNA sequences.

3. Cell Culture Systems/Cell Culture Vectors

Propogation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (see Tissue Culture, Academic Press,Kruse and Patterson eds, 1973). Employed herein was the COS-7 line ofmonkey kidney fibroblasts as the host for the production of immuneinterferon (25a). However, the experiments detailed here could beperformed in any cell line which is capable of the replication andexpression of a compatible vector, e.g., WI38, BHK, 3T3, CHO, VERO, andHeLa cell lines. Additionally, what is required of the expression vectoris an origin of replication and a promoter located in front of the geneto be expressed, along with any necessary ribosome binding sites, RNAsplice sites, polyadenylation site, and transcriptional terminatorsequences. While these essential elements of SV40 have been exploitedherein, it will be understood that the invention, although describedherein in terms of a preferred embodiment, should not be construed aslimited to these sequences. For example, the origin of replication ofother viral (e.g., Polyoma, Adeno, VSV, BPV, and so forth) vectors couldbe used, as well as cellular origins of DNA replication which couldfunction in a nonintegrated state.

B. Vector Systems

1. Direct Expression of Mature Immune Interferon in E. coli

The procedure used to obtain direct expression of IFN-γ in E. coli as amature interferon polypeptide (minus signal sequence) was a variant ofthat employed earlier for human growth hormone (26) and human leukocyteinterferon (1), insofar as it involved the combination of synthetic(N-terminal) and cDNAs.

As deduced from the nucleotide sequence of p69, described infra, and bycomparison with the known cleavage site between signal peptide andmature polypeptide for several IFN-αs (2), IFN-γ has a hydrophobicsignal peptide of 20 amino acids followed by 146 amino acids of matureIFN-γ (FIG. 5). As shown in FIG. 7, a BstNI restriction endonucleasesite is conveniently located at amino acid 4 of mature IFN-γ. Twosynthetic oligodeoxynucleotides were designed which incorporate an ATGtranslational initiation codon, codons for amino acids 1, 2 and 3(cysteine-tyrosine-cysteine) and create an EcoRI cohesive end. Thesedeoxyoligonucleotides were ligated to a 100 base pair BstNI-PstIfragment of p69 to construct a 1115 base pair synthetic-natural hybridgene which codes for IFN-γ and which is bounded by EcoRI and PstIrestriction sites. This gene was inserted into the plasmid pLeIF A trp103 between the EcoRI and PstI sites to give the expression plasmidpIFN-γ trp 48. In this plasmid the IFN-γ gene is expressed under thecontrol of the E. coli trp promoter. (pLeIF A trp 103 is a derivative ofpLeIF A 25 in which the EcoRI site distal to the LeIF A gene wasremoved. The procedure used to remove this EcoRI site has been describedpreviously (27)).

2. Expression in Yeast

To express a heterologous gene such as the cDNA for immune interferon inyeast, it was necessary to construct a plasmid vector containing fourcomponents. The first component is the part which allows fortransformation of both E. coli and yeast and thus must contain aselectable gene from each organism. (In this case, this is the gene forampicillin resistance from E. coli and the gene TRP1 from yeast.) Thiscomponent also requires an origin of replication from both organisms tobe maintained as a plasmid DNA in both organisms. (In this case, this isthe E. coli origin from pBR322 and the ars1 origin from chromosome IIIof yeast.)

The second component of the plasmid is a 5'-flanking sequence from ahighly expressed yeast gene to promote transcription of adownstream-placed structural gene. In this case, the 5'-flankingsequence used is that from the yeast 3-phosphoglycerate kinase (PGK)gene. The fragment was constructed in such a way so as to remove the ATGof the PGK structural sequence as well as 8 bp upstream from this ATG.This sequence was replaced with a sequence containing both an XbaI andEcoRI restriction site for convenient attachment of this 5'-flankingsequence to the structural gene.

The third component of the system is a structural gene constructed insuch a manner that it contains both an ATG translational start andtranslational stop signals. The isolation and construction of such agene is described infra.

The fourth component is a yeast DNA sequence containing the 3'-flankingsequence of a yeast gene, which contains the proper signals fortranscription termination and polyadenylation.

With all these components present, immune interferon has been producedin yeast.

3. Expression in Mammalian Cell Culture

The strategy for the synthesis of immune interferon in mammalian cellculture relied on the development of a vector capable of both autonomousreplication and expression of a foreign gene under the control of aheterologous transcriptional unit. The replication of this vector intissue culture was accomplished by providing a DNA replication origin(derived from SV40 virus), and providing helper function (T antigen) bythe introduction of the vector into a cell line endogenously expressingthis antigen (28, 29). The late promoter of SV40 virus preceded thestructural gene of interferon and ensured the transcription of the gene.

The vector used to obtain expression of IFN-γ consisted of pBR322sequences which provided a selectable marker for selection in E. coli(ampicillin resistance) as well as an E. coli origin of DNA replication.These sequences were derived from the plasmid pML-1 (28) and encompassedthe region spanning the EcoRI and BamHI restriction sites. The SV40origin is derived from a 342 base pair PvuII-HindIII fragmentencompassing this region (30, 31) (both ends being converted to EcoRIends). These sequences, in addition to comprising the viral origin ofDNA replication, encode the promoter for both the early and lasttranscriptional unit. The orientation of the SV40 origin region was suchthat the promoter for the late transcriptional unit was positionedproximal to the gene encoding interferon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a sucrose gradient centrifugation of induced PeripheralBlood Lymphocyte (PBL) Poly(A)+ RNA. Two peaks of interferon activitywere observed (as shown by the hatched boxes) with sizes of 12S and 16S.The positions of ribosomal RNA markers (centrifuged independently) arelabeled above the absorbance profile.

FIG. 2 depicts an electrophoresis of induced PBL Poly(A)+ RNA through anacid-urea-agarose. Only one peak of activity was observed, whichcomigrated with 18S RNA. The positions of ribosomal RNA markers whichwere electrophoresed in an adjacent lane and visualized by ethidiumbromide staining are labeled above the activity profile.

FIG. 3 shows hybridization patterns of 96 colonies with induced anduninduced ³² P-labelled cDNA probes. 96 individual transformants weregrown in a microtiter plate, replica plated on two nitrocellulosemembranes, and then the filters were hybridized with ³² P-cDNA probesprepared from either induced mRNA (above) or mRNA isolated fromuninduced PBL cultures (uninduced, below). The filters were washed toremove non-hybridized RNA and then exposed to X-ray film. This set offilters is representative of 86 such sets (8300 independent colonies).An example of an "induced" clone is labelled as H12.

FIG. 4 is a restriction endonuclease map of the clone 69 cDNA insert.The cDNA insert is bounded by PstI sites (dots at both ends) and oligodC-dG tails (single lines). The number and size of fragments produced byrestriction nuclease cleavage was estimated by electrophoresis through 6percent acrylamide gels. Positions of sites was confirmed by nucleicacid sequencing (presented in FIG. 5). The coding region of the largestopen reading frame is boxed and the hatched region represents theputative 20 residue signal peptide sequence, while the stipled regionrepresents the mature IIF sequence (146 amino acids). The 5' end of themRNA is to the left while the 3' end is to the right.

FIG. 5 illustrates the nucleotide sequence of the plasmid p69 cDNAinsert. The deduced amino acid sequence of the longest open readingframe is also presented. The putative signal sequence is represented bythe residue labelled S1 to S20.

FIG. 6 is a comparison of IFN-γ mRNA structure with that of leukocyte(IFN-α) and fibroblast (IFN-β) interferons. The clone 69 mRNA (labelledimmune) contains significantly greater amounts of untranslatedsequences.

FIG. 7 is a schematic diagram of the construction of the IFN-γexpression plasmid pIFN-γ trp 48. The starting material is the 1250 basepair PstI cDNA insert from plasmid p69.

FIG. 8 shows a diagram of plasmid used for expression of IFN-γ in monkeycells.

FIG. 9 depicts a Southern hybridization of eight different EcoRIdigested human genomic DNAs hybridized with a ³² P-labelled 600 basepair DdeI fragment from the cDNA insert of p69. Two EcoRI fragmentsclearly hybridize with the probe in each DNA sample.

FIG. 10 depicts a Southern hybridization of human genomic DNA digestedwith six different restriction endonucleases hybridized with the ³²P-labelled probe from p69.

FIG. 11 schematically illustrates the restriction map of the 3.1 kbpHindIII insert of vector pB1 from which the PGK promoter was isolated.Indicated is the insertion of an EcoRI site and an XbaI site in the5'-flanking DNA of the PGK gene.

FIG. 12 illustrates the 5'-flanking sequence plus the initial codingsequence for the PGK gene before insertion of an XbaI and EcoRI sites.

FIG. 13 schematically illustrates techniques used to insert an XbaI siteat position--8 in the PGK promoter and to isolate a 39 bp fragment ofthe 5'-flanking sequence of PGK containing this XbaI end and a Sau3Aend.

FIG. 14 schematically illustrates the construction of a 300 bp fragmentcontaining the above 39 bp fragment, additional PGK 5'-flanking sequence(265 bp) from PvuI to Sau3A (see FIG. 11), and a EcoRI site adjacent toXbaI.

FIG. 15 schematically illustrates the construction of the 1500 bp PGKpromoter fragment (HindIII/EcoRI) which contains, in addition to thefragment constructed in FIG. 14, a 1300 bp HindIII to PvuI fragment fromPGK 5'-flanking sequence (see FIG. 11).

FIG. 16 illustrates the composition of an expression vector for humanimmune interferon in yeast, containing the modified PGK promoter, theIFN-γ cDNA and the terminator region of the yeast PGK gene as describedin more detail herein.

DETAILED DESCRIPTION A. Source of IFN-γ mRNA

Peripheral Blood Lymphocytes (PBLs) were derived from human donors byleukophoresis. PBLs were further purified by Ficoll-Hypaque gradientcentrifugation and then cultured at a concentration of 5×10⁶ cells/ml inRPMI 1640, 1 percent L-glutamine, 25 mM HEPES, and 1 percentpenicillin-streptomycin solution (Gibco, Grand Island, NY). These cellswere induced to produce IFN-γ by the mitogen staphlococcal enterotoxin B(1 μg/ml) and cultured for 24 to 48 hours at 37° C. in 5 percent CO₂.Desacetylthymosin-α-1 (0.1 μg/ml) was added to PBL cultures to increasethe relative yield of IFN-γ activity.

B. Messenger RNA Isolation

Total RNA from PBL cultures was extracted essentially as reported byBerger, S. L. et al. (33). Cells were pelleted by centrifugation andthen resuspended in 10 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl₂and 10 mM ribonucleoside vanadyl complex. Cells were lysed by theaddition of NP-40 (1 percent final concentration), and nuclei werepelleted by centrifugation. The supernatant contained the total RNAwhich was further purified by multiple phenol and chloroformextractions. The aqueous phase was made 0.2M in NaCl and then total RNAwas precipitated by the addition of two volumes of ethanol. RNA fromuninduced (nonstimulated) cultures was isolated by the same methods.Oligo-dT cellulose chromatography was utilized to purify mRNA from thetotal RNA preparations (34). Typical yields from 1-2 liters of culturedPBLs were 5-10 milligrams of total RNA and 50-200 micrograms of Poly(A)+RNA.

C. Size Fractionation of mRNA

Two methods were used to fractionate mRNA preparations. These methodswere used independently (rather than in unison) and each resulted in asignificant enrichment of IFN-γ mRNA.

Sucrose gradient centrifugation in the presence of the denaturantformamide was used to fractionate mRNA. Gradients of 5 percent to 25percent sucrose in 70 percent formamide (32) were centrifuged at154,000×g for 19 hours at 20° C. Successive fractions (0.5 ml) were thenremoved from the top of the gradient, ethanol precipitated, and analiquot was injected into Xenopus laevis oocytes for translation of themRNA (35). After 24 hrs. at room temperature, the incubation medium wasthen assayed for antiviral activity in a standard cytopathic effectinhibition assay employing Vesicular Stomatitis Virus (Indiana strain)or Encephalomyocarditis Virus on WISH (human amnion) cells as describedby Stewart (36), except that the samples were incubated with the cellsfor 24 hours (instead of 4) prior to challenge with the virus. Twoactivity peaks were consistently observed in sucrose gradientfractionated RNA (FIG. 1). One peak sedimented with a calculated size of12S and contained 100-400 units/ml of antiviral activity (compared witha IFN-α standard) per microgram of RNA injected. The other peak ofactivity sedimented as 16S in size and contained about half the activityof the slower sedimenting peak. Each of these activity peaks appears tobe due to IFN-γ, since no activity was observed when the same fractionswere assayed on a bovine cell line (MDBK) which is not protected byhuman IFN-γ. Both IFN-α activity and IFN-β activity would have beeneasily detected with the MDBK assay (5).

Fractionation of mRNA (200 μg) was also performed by electrophoresisthrough acid urea agarose gels. The slab agarose gel (37, 38) wascomposed of 1.75 percent agarose, 0.025M sodium citrate, pH 3.8 and 6Murea. Electrophoresis was performed for 7 hours at 25 milliamp and 4° C.The gel was then fractionated with a razor blade. The individual sliceswere melted at 70° C. and extracted twice with phenol and once withchloroform. Fractions were then ethanol precipitated and subsequentlyassayed for IFN-γ mRNA by injection into Xenopus laevis oocytes andantiviral assay. Only one peak of activity was observed in gelfractionated samples (FIG. 2). This peak comigrated with 18S RNA and hadan activity of 600 units/ml per microgram of injected RNA. This activityalso appeared to be IFN-γ specific, since it did not protect MDBK cells.

The size discrepancy between activity peaks observed on sucrosegradients (12S and 16S) and acid urea gels (18S) may be explained by theobservation that these independent fractionation methods are notperformed under total denaturing conditions.

D. Preparation of a Colony Library Containing IFN-γ Sequences

3 μg of gel-fractionated mRNA was used for the preparation of doublestranded cDNA by standard procedures (26, 39). The cDNA was sizefractionated on a 6 percent polyacrylamide gel. Two size fractions wereelectroeluted, 800-1500 bp (138 ng) and 22 1500 bp (204 ng). 35 ngportions of each size cDNA was extended with deoxyC residues usingterminal deoxynucleotidyl transferase (40) and annealed with 300 ng ofthe plasmid pBR322 (41) which had been similarly tailed with deoxyGresidues at the PstI site (40). Each annealed mixture was thentransformed into E. coli K12 strain 294. Approximately 8000transformants were obtained with the 800-1500 bp cDNA and 400transformants were obtained with the >1500 bp cDNA.

E. Screening of Colony Library for Induced cDNAs

The colonies were individually inoculated into wells of microtitreplates containing LB (58)+5 μg/ml tetracycline and stored at -20° C.after addition of DMSO to 7 percent. Two copies of the colony librarywere grown up on nitrocellulose filters and the DNA from each colonyfixed to the filter by the Grunstein-Hogness procedure (42).

³² P-labelled cDNA probes were prepared using 18S size gel fractionatedmRNA from induced and uninduced PBL cultures. Oligo dT₁₂₋₁₈ was theprimer and reaction conditions have been previously described (1).Filters containing 8000 transformants from the 600-1500 bp cDNA size cutand 400 transformants from the >1500 bp cDNA size cut were hybridizedwith 20×10⁶ cpm of induced ³² P-cDNA. A duplicate set of filters washyrbidized with 20×10⁶ cpm of uninduced ³² P-cDNA. Hybridization was for16 hours using conditions described by Fritsch et al. (43). Filters wereextensively washed (43) and then exposed to Kodak XR-5 X-ray film withDuPont Lightning-Plus intensifying screens for 16-48 hours. Eachcolony's hybridization pattern with the two probes was compared.Approximately 40 percent of the colonies clearly hybridized with bothprobes, while approximately 50 percent of the colonies failed tohybridize with either probe (presented in FIG. 3). 124 colonieshybridized significantly with the induced probe but undetectably or moreweakly with the uninduced probe. These colonies were individuallyinoculated into wells of microtitre plates, grown and transferred tonitrocellulose filters, and hybridized with the same two probes, asdescribed above. Plasmid DNA isolated from each of these colonies by arapid method (44) was also bound to nitrocellulose filters andhybridized (45) with the induced and uninduced probes. DNA from 22colonies hybridized with only the induced probe and were termed"induced" colonies.

F. Characterization of Induced Colonies

Plasmid DNA was prepared from 5 of the induced colonies (46) and usedfor characterization of the cDNA inserts. Restriction endonucleasemapping of five induced plasmids (p67, p68, p69, p71 and p72) suggestedthat four had similar restriction nuclease maps. These four (p67, p69,p71 and p72) each had four DdeI sites, 2 HinfI sites, and a single RsaIsite in the cDNA insert. The fifth plasmid (p68 ) contained a commonDdeI fragment and appeared to be a short cDNA clone related to the otherfour. The homology suggested by restriction nuclease mapping wasconfirmed by hybridization. A ³² P-labelled DNA probe was prepared (47)from a 600 bp DdeI fragment of the p67 plasmid and used forhybridization (42) to the other induced colonies. All five of therestriction nuclease mapped colonies cross-hybridized with this probe,as did 17 other colonies of the 124 chosen in the induced/uninducedscreening. The length of cDNA insert in each of these cross-hybridizingplasmids was determined by PstI digestion and gel electrophoresis. Theclone with the longest cDNA insert appeared to be clone 69 with aninsert length of 1200-1400 bp. This DNA was used for all furtherexperiments, and its restriction endonuclease map is shown in FIG. 4.

The cDNA insert in p69 was demonstrated to be IFN-γ cDNA by itsexpression products, produced in three independent expression systems,yielding antiviral activity, as described in more detail infra.

G. Sequence Analysis of cDNA Insert of p69

The complete nucleotide sequence of the plasmid p69 cDNA insert wasdetermined by the dideoxynucleotide chain termination method (48) aftersubcloning fragments into the M13 vector mp7 (49) and by theMaxam-Gilbert chemical procedure (52). The longest open reading frameencodes a protein of 166 amino acids, presented in FIG. 5. The firstresidue encoded is the first met codon encountered in the 5' end of thecDNA. The first 20 residues at the amino terminus probably serves as asignal sequence for the secretion of the remaining 146 amino acids. Thisputative signal sequence has features in common with other characterizedsignal sequences such as size and hydrophobicity. Furthermore, the fouramino acids found at the putative cleavage sequence (ser-leu-gly-cys)are identical with four residues found at the cleavage point of severalleukocyte interferons (LeIF B, C, D, F, and H, (2)). The encoded matureamino acid sequence of 146 amino acids (hereinafter referred to as"recombinant human immune interferon") has a molecular weight of 17,140.

There are two potential glycosylation positions (50) in the encodedprotein sequence, at amino acids 28 to 30 (asn-gly-thr) and amino acids100 to 102 (asn-tyr-ser). The existence of these positions is consistentwith the observed glucosylation of human IFN-γ (6, 51). In addition, theonly two cysteine residues (positions 1 and 3) are sterically too closeto form a disulfide bridge, which is consistent with the observedstability of IFN-γ in the presence of reducing agents such asβ-mercaptoethanol (51). The deduced mature amino acid sequence isgenerally quite basic, with 30 total lysine, arginine, and histidineresidues and only 19 total aspartic acid and glutamic acid residues.

The mRNA structure of IFN-γ as deduced from DNA sequence of plasmid p69is distinctively different from IFN-α (1, 2) or IFN-β (5) mRNA. Aspresented in FIG. 6, the coding region of IFN-γ is shorter while the 5'untranslated and 3' untranslated regions are much longer than eitherIFN-α or IFN-β.

H. Expression of Recombinant Human Immune Interferon in E. coli

With reference to FIG. 7, 50 μg of plasmid p69 were digested with PstIand the 1250 base pair insert isolated by gel electrophoresis on a 6percent polyacrylamide gel. Approximately 10 μg of this insert waselectroeluted from the gel. 5 μg of this PstI fragment was partiallydigested with 3 units of BstNI (Bethesda Research Labs) for 15 minutesat 37° C. and the reaction mixture purified on a 6 percentpolyacrylamide gel. Approximately 0.5 μg of the desired 1100 base pairBstNI-PstI fragment was recovered. The two indicateddeoxyoligonucleotides, 5'-dAATTCATGTGTTATTGTC and 5'-dTGACAATAACACATG(FIG. 7) were synthesized by the phosphotriester method (53) andphosphorylated as follows. 100 pmoles of each deoxyoligonucleotide werecombined in 30 μl of 60 mM Tris-HCl (pH 8), 10 mM MgCl₂, 15 mMβ-mercaptoethanol and 240 μCi (γ-³² P)ATP (Amersham, 5000 Ci/mmole). 12units of T4 polynucleotide kinase were added and the reaction allowed toproceed at 37° C. for 30 minutes. 1 μl of 10 mM ATP wa added and thereaction allowed to proceed an additional 20 minutes. After φ-OH/CHCl₃extraction the oligomers were combined with 0.25 μg of the BstNI-PstI1100 base pair fragment and ethanol precipitated. These fragments wereligated at 20° C. for 2 hours in 30 μl of 20 mM Tris-HCl (pH 7.5), 10 mMMgCl₂, 10 mM dithiothreitol, 0.5 mM ATP and 10 units T4 DNA ligase. Themixture was digested for 1 hour with 30 units of PstI and 30 units ofEcoRI (to eliminate polymerization through ligation of cohesive termini)and electrophoresed on a 6 percent polyacrylamide gel. The 1115 basepair product (110,000 cpm) was recovered by electroelution.

The plasmid pLeIF A trp 103 (FIG. 7) is a derivative of the plasmidpLeIF A 25 (1) in which the EcoRI site distal to the LeIF A gene hasbeen removed (27). 3 μg of pLeIF A trp 103 was digested with 20 units ofEcoRI and 20 units of PstI for 90 minutes at 37° C. and electrophoresedon a 6 percent polyacrylamide gel. The large (˜3900 base pair) vectorfragment was recovered by electroelution. The 1115 base pair EcoRI-PstIIFN-γ DNA fragment was ligated into 0.15 μg of this prepared vector.Transformation of E. coli K-12 strain 294 (ATCC No. 31446) gave 120tetracycline resistant colonies. Plasmid DNA was prepared from 60 ofthese transformants and digested with EcORI and PstI. Three of theseplasmids contained the desired 1115 base pair EcoRI-PstI fragment. DNAsequence analysis verified that these plasmids had the desirednucleotide sequence at the junctions between the trp promoter, syntheticDNA and cDNA. One of these plasmids pIFN-γ trp 48 was chosen foradditional study. This plasmid was used to transform the E. coli K-12strain W3110 (ATCC No. 27325).

I. Gene Structure of the IFN-γ Coding Sequence

The structure of the gene coding for IFN-γ was analyzed by Southernhybridization. In this procedure (54), 5 micrograms of high molecularweight human lymphocyte DNA (prepared as in 55) is digested tocompletion with various restriction endonucleases, electrophoresed on1.0 percent agarose gels (56), and blotted to a nitrocellulose filter(54). A ³² P-labelled DNA probe was prepared (47) from a 600 bp DdeIfragment of the cDNA insert of p69 and hybridized (43) with thenitrocellulose-DNA blot. 10⁷ counts per minute of the probe werehybridized for 16 hours and then washed as described (43). Eight genomicDNA samples from different human donors were digested with the EcoRIrestriction endonuclease and hybridized with the p69 ³² P-labelledprobe. As presented in FIG. 9, two clear hybridization signals areobserved with sizes of 8.8 kilobase pairs (kbp) and 2.0 kbp as estimatedby comparison of mobilities with HindIII digested λDNA. This could bethe result of two IFN-γ genes or a single gene split by an EcoRI site.Since the p69 cDNA contains no EcoRI site, an intervening sequence(intron) with an internal EcoRI site would be necessary to explain asingle gene. To distinguish between these possibilities, anotherSouthern hybridization was performed with the same probe against fiveother endonuclease digestions of a single human DNA (FIG. 10). Twohybridizing DNA fragments were observed with two other endonucleasedigests, PvuII (6.7 kbp and 4.0 kbp) and HincII (2.5 kbp and 2.2 kbp).However, three endonuclease digestion patterns provide only a singlehybridizing DNA fragment: HindIII (9.0 kbp), BglII (11.5 kbp) and BamHI(9.5 kbp). Two IFN-γ genes would have to be linked at an unusually closedistance (less than 9.0 kbp) to be contained within the same HindIIIhybridizing fragment. This result suggests that only a single homologousIFN-γ gene (unlike the many related IFN-α genes) is present in humangenomic DNA and that this gene is split by one or more intronscontaining EcoRI, PvuII, and HincII sites. This prediction was supportedby hybridization of a ³² P-labelled (47) fragment prepared from just the3' untranslated region of the cDNA from p69 (130 bp DdeI fragment from860 bp to 990 bp in FIG. 5) against an EcoRI digest of human genomicDNA. Only the 2.0 kbp EcoRI fragment hybridized to this probe,suggesting that this fragment contains the 3' untranslated sequences,while the 8.8 kbp EcoRI fragment contains the 5' sequences. The genestructure of IFN-γ (one gene with at least one intron) is distinctlydifferent from IFN-α (multiple genes (2) without introns (56)) or IFN-β(one gene with no introns (57)).

J. Preparation of Bacterial Extracts

An overnight culture of E. coli W3110/pIFN-γ trp 48 in Luria broth+5micrograms per ml tetracycline was used to inoculate M9 (58) mediumcontaining 0.2 percent glucose, 0.5 percent casamino acids, and 5micrograms per ml tetracycline at a 1:100 dilution. Indole acrylic acidwas added to a final concentration of 20 micrograms per ml when A₅₅₀ wasbetween 0.1 and 0.2. Ten ml samples were harvested by centrifugation atA₅₅₀ =1.0 and resuspended immediately in 1 ml phosphate buffered salinecontaining 1 mg per ml bovine serum albumin (PBS-BSA). Cells were openedby sonication and cleared of debris by centrifugation. The supernatantswere stored at 4° C. until assay. Interferon activity in thesupernatants was determined to be 250 units/ml by comparison with IFN-αstandards by the cytopathic effect (CPE) inhibition assay.

K. Transformation of Yeast/Strains and Media

Yeast strains were transformed as previously described (59). E. colistrain JA300 (thr leuB6 thi thyA trpC1117 hsdm⁻ hsdR⁻⁰ str^(R)) (20) wasused to select for plasmids containing functional TRPI gene. Yeaststrain RH218 having the genotype (a trp1 gal2 SUC2 mal CUPI) (18) wasused as yeast transformation host. RH218 has been deposited withoutrestriction in the American Type Culture Collection, ATCC No. 44076. M9(minimal medium) with 0.25 percent casamino acids (CAA) and LB (richmedium) were as described by Miller (58) with the addition of 20 μg/mlampicillin (Sigma) after media is autoclaved and cooled. Yeast weregrown on the following media: YEPD contained 1 percent yeast extract, 2percent peptone and 2 percent glucose ±3 percent Difco agar. YNB+CAAcontained 6.7 grams of yeast nitrogen base (without amino acids) (YNB)(Difco), 10 mg of adenine, 10 mg of uracil, 5 grams CAA, 20 gramsglucose and ±30 grams agar per liter.

L. Construction of Yeast Expression Vector

1. 10 μg of YRp7 (14, 15, 16) was digested with EcoRI. Resulting stickyDNA ends were made blunt using DNA Polymerase I (Klenow fragment).Vector and insert were run on 1 percent agarose (SeaKem) gel, cut fromthe gel, electroeluted and extracted 2× with equal volumes of chloroformand phenol before precipitation with ethanol. The resulting blunt endDNA molecules were then ligated together in a final volume of 50 μl for12 hours at 12° C. This ligation mix was then used to transform E. colistrain JA300 to ampicillin resistance and tryptophan prototrophy.Plasmids containing the TRPI gene in both orientations were isolated.pFRW1 had the TRPI gene in the same orientation as YRp7 while pFRW2 hadthe TRPI gene in the opposite orientation.

20 μg of pFRW2 was linearized with HindIII and electrophoresed on a 1percent agarose gel. Linear molecules were eluted from the gel and 200ng were then ligated with 500 ng of the 3.1 kb HindIII insert of plasmidpB1 (13) which is a restriction fragment containing the yeast3-phosphoglycerate kinase gene. The ligation mix was used to transformE. coli strain 294 to ampicillin resistance and tetracyclinesensitivity. Plasmid prepared from one such recombinant had an intactTRP1 gene with the 3.1 kbp HindIII fragment from pB1 inert DNA in theHindIII site of the tetracycline resistance gene. This plasmid ispFRM31. 5 μg of pFRM31 was completely digested with EcoRI, extractedtwice with phenol and chloroform then ethanol precipitated. The cohesiveends of the molecule were filled in using DNA Polymerase I (Klenowfragment) in a reaction which was made 250 μM in each deoxynucleosidetriphosphate. The reaction was performed for 20 minutes at 14° C. atwhich time the DNA was extracted two times with phenol-chloroform, andthen precipitated with ethanol. The resuspended DNA was then completelydigested with ClaI and electrophoresed on a 6 percent acrylamide gel.The vector fragment was eluted from the gel, phenol-chloroform extractedand ethanol precipitated.

The six N-terminal amino acids of the 3-phosphoglycerate kinase enzymepurified from humans are as follows: ##STR1##

One of the translational reading frames generated from the DNA sequenceof the 141 bp Sau3A-to-Sau3A restriction fragment (containing theinternal HincII site; see PGK restriction map FIG. 11) produces thefollowing amino acid sequence. ##STR2##

After removal of initiator methionine, it is seen that PGK N-terminalamino acid sequence has 5 of 6 amino acid homology with N-terminal aminoacid sequence of human PGK.

This sequencing result suggested that the start of the yeast PGKstructural gene is coded for by DNA in the 141 bp Sau3A restrictionfragment of pB1. Previous work (20) has suggested that the DNA sequencesspecifying the PGK mRNA may reside in this area of the HindIII fragment.Further sequencing of the 141 bp Sau3A fragment gives more DNA sequenceof the PGK promoter (FIG. 12).

A synthetic oligonucleotide with the sequence 5'ATTTGTTGTAAA3' wassynthesized by standard methods (Crea et al., Nucleic Acids Res. 8, 2331(1980)). 100 ng of this primer was labeled at the 5' end using 10 unitsof T4 polynucleotide kinase in a 20 μl reaction also containing 200 μCiof [γ³² -P] ATP. This labeled primer solution was used in aprimer-repair reaction designed to be the first step in a multi-stepprocess to put an EcoRI restriction site in the PGK 5'-flanking DNA justpreceding PGK structure gene sequence.

100 μg of pB1 (20) was completely digested with HaeIII then run on a 6percent polyacrylamide gel. The uppermost band on the ethidum stainedgel (containing PGK promoter region) was isolated by electroelution asdescribed above. This 1200 bp HaeIII piece of DNA was restricted withHincII then run on a 6 percent acrylamide gel. The 650 bp band wasisolated by electroelution. 5 μg of DNA was isolated. This 650 bpHaeIII-to-HincII piece of DNA was resuspended in 20 μl H₂ O, then mixedwith the 20 μl of the phosphorylated primer solution described above.This mixture was 1× phenol-chloroform extracted then ethanolprecipitated. Dried DNA was resuspended in 50 μl of H₂ O and then heatedin a boiling water bath for seven minutes. This solution was thenquickly chilled in a dry ice-ethanol bath (10-20 seconds) thentransferred to an ice-water bath. To this solution was added 50 μl of asolution containing 10 μl of 10× DNA polymerase I buffer (BoehringerMannheim), 10 μl of a solution previously made 2.5 mM in eachdeoxynucleoside triphosphate (dATP, dTTP, dGTP and dCTP), 25 μl of H₂ Oand 5 units of DNA Polymerase I, Klenow fragment. This 100 μl reactionwas incubated at 37° C. for 4 hours. The solution was then 1×phenol-chloroform extracted, ethanol precipitated, dried bylyophilization then exhaustively restricted with 10 units of Sau3A. Thissolution was then run on a 6 percent acrylamide gel. The bandcorresponding to 39 bp in size was cut from the gel then isolated byelectroelution described above. This 39 bp band has one blunt end andone Sau3A sticky end. This fragment was cloned into a modified pFIF trp69 vector (5). 10 μg of pFIF trp 69 was linearized with XbaI, 1× phenolchloroform extracted, then ethanol precipitated. The XbaI sticky end wasfilled in using DNA Polymerase I Klenow fragment in a 50 μl reactioncontaining 250 μM in each nucleoside triphosphate. This DNA was cut withBamHI then run on a 6 percent acrylamide gel. The vector fragment wasisolated from the gel by electroelution then resuspended in 20 μl H₂ O.20 ng of this vector was ligated with 20 ng of the 39 bp fragmentprepared above for 4 hours at room temperature. One-fifth of theligation mix was used to transform E. coli strain 294 to ampicillinresistance (on LB +20 μg/ml amp plates. Plasmids from the transformantswere examined by a quick screen procedure (44). One plasmid, pPGK-39 wasselected for sequence analysis. 20 μg of this plasmid was digested withXbaI, ethanol precipitated then treated with 1000 units of bacterialalkaline phosphase at 68° C. for 45 min. The DNA was 3×phenol-chloroform extracted, then ethanol precipitated. Thedephosphorylated ends were then labeled in a 20 μl reaction containing200 μCi of [γ³² -P] ATP and 10 units of T₄ polynucleotide kinase. Theplasmid was cut with SalI and run on a 6 percent acrylamide gel.

The labeled insert band was isolated from the gel and sequenced by thechemical degradation method (52). The DNA sequence at the 3'-end of thispromoter piece was as expected.

2. Construction of 312 bp PvuI-to-EcoRI PGK Promoter Fragment

25 μg of pPGK-39 (FIG. 13) was simultaneously digested with SalI andXbaI (5units each) then electrophoresed on a 6 percent gel. The 390 bpband countaining the 39 bp promoter piece was isolated byelectroelution. The resuspended DNA was restricted with Sau3A thenelectrophoresed on an 8 percent acrylamide gel. The 39 bp PGK promoterband was isolated by electroelution. This DNA contained 39 bp of the 5'end of the PGK promoter on a Sau3A-to-XbaI fragment.

25 μg of pB1 was restricted with PvuI and KpnI then electrophoresed on a6 percent acrylamide gel. The 0.8 kbp band of DNA was isolated byelectroelution, then restricted with Sau3A and electrophoresed on a 6percent acrylamide gel. The 265 bp band from the PGK promoter (FIG. 11)was isolated by electroelution.

This DNA was then ligated with the 39 bp promoter fragment from abovefor two hours at room temperature. The ligation mix was restricted withXbaI and PvuI then electrophoresed on a 6 percent acrylamide gel. The312 bp Xba-to-PvuI restriction fragment was isolated by electroelution,then added to a ligation mix containing 200 ng of pBR322 (41)(previously isolated missing the 162 bp PvuI-to-PstI restrictionfragment) and 200 ng of the XbaI-to-PstI LeIF A cDNA gene previouslyisolated from 20 μg of pLeIF trp A 25. This three-factor-ligation mixwas used to transform E. coli strain 294 to tetracycline resistance.Transformant clonies were miniscreened (44) and one of the colonies,pPGK-300 was isolated as having 304 bp of PGK 5'-flanking DNA fused tothe LeIF A gene in a pBR322 based vector. The 5' end of the LeIF A genehas the following sequence: 5'-CTAGAATTC-3'. Thus fusion of the XbaIsite from the PGK promoter fragment into this sequence allows for theaddition to the XbaI site an EcoRI site. pPGK-300 thus contains part ofthe PGK promoter isolated in a PvuI-to-EcoRI fragment.

3. Construction of a 1500 bp EcoRI-to-EcoRI PGK Promoter Fragment

10 μg of pB1 was digested with PvuI and EcoRI and run on a 6 percentacrylamide gel. The 1.3 kb PvuI-to-EcoRI DNA band from the PGK5'-flanking DNA was isolated by electroelution. 10 μg of pPGK-300 wasdigested with EcoRI and PvuI and the 312 bp promoter fragment wasisolated by electroelution after electrophoresing the digestion mix on a6 percent acrylamide gel. 5 μg of pFRL4 was cut with EcoRI, ethanolprecipitated then treated with bacterial alkaline phosphatase at 68° C.for 45 minutes. After three extractions of DNA with phenol/chloroform,ethanol precipitation, and resuspension in 20 ml of H₂ O; 200 ng of thevector was ligated with 100 ng of 312 by EcoRI-to-PvuI DNA from pPGK-300and 100 ng of EcoRI-to-PvuI DNA from pB1. The ligation mix was used totransform E. coli strain 294 to ampicillin resistance. One of thetransformants obtained was pPGK- 1500. This plasmid contains the 1500 bpPGK promoter fragment as an EcoRI-to-EcoRI or HindIII-to-EcoRI piece ofDNA.

10 μg of pPGK-1500 was completely digested with ClaI and EcoRI then thedigestion mix was electrophoresed on a 6 percent acrylamide gel. The 900bp fragment containing the PGK promoter was isolated by electroelution.10 μg of pIFN-γ trp 48 was completely digested with EcoRI and HincII andelectrophoresed on a 6 percent acrylamide gel. The 938 bp bandcontaining the directly expressable IFN-γ cDNA was isolated from the gelby electroelution.

The yeast expression vector was constructed in a three factor reactionby ligating together the PGK promoter fragment (on a ClaI-to-EcoRIpiece), the deleted pFRM-31 and the above isolated IFN-γ cDNA. Theligation reaction was incubated at 14° C. for 12 hours. The ligation mixwas then used to transform E. coli strain 294 to ampicillin resistance.Transformants were analyzed for the presence of the properly constructedexpression plasmid, pPGK-IFN-γ (FIG. 16). Plasmids containing theexpression system were used to transform spheroplasts of yeast strainRH218 to tryptophan prototropy in agar missing tryptophan. Theserecombinant yeast were then assayed for the presence of recombinanthuman immune interferon.

Yeast extracts were prepared as follows: Ten ml cultures were grown inYNB+CAA until reaching A₆₆₀ ≃1-2, collected by centrifugation thenresuspended in 500 μl PBS buffer (20 mM NaH₂ PO₄, pH=7.4, 150 mM NaCl).An equal volume of glass beads (0.45-0.5 mm) were added and the mixturewas then vortexed for 2'. The extracts were spun 30 seconds at 14,000rpm and supernatant removed: Interferon activity in the supernatant wasdetermined to be 16,000 units/ml by comparison with IFN-α standard usingthe CPE inhibition assay.

M. Construction of Cell Culture Vector pSVγ69

The 342 base pair HindIII-PvuII fragment encompassing the SV40 originwas converted to an EcoRI restriction site bound fragment. The HindIIIsite was converted by the addition of a synthetic oligomer(5'dAGCTGAATTC) and the PvuII site was converted by blunt-end ligationinto an EcoRI site filled in using Polymerase I (Klenow fragment). Theresulting EcoRI fragment was inserted into the EcoRI site of pML-1 (28).A plasmid with the SV40 late promoter oriented away from the amp^(R)gene was further modified by removing the EcoRI site nearest the amp^(R)gene of pML-1 (27).

The 1023 base pair HpaI-BglII fragment of cloned HBV DNA (60) wasisolated and the HpaI site of hepatitis B virus (HBV) converted to anEcoRI site with a synthetic oligomer (5'dGCGAATTCGC). This EcoRI-BglIIbounded fragment was directly cloned into the EcoRI-BamHI sites of theplasmid described above carrying the origin of SV40.

Into the remaining EcoRI site was inserted the IFN-γ gene on a 1250 basepair PstI fragment of p69 after conversion of the PstI ends to EcoRIends. Clones were isolated in which the SV40 late promoter preceded thestructural gene of IFN-γ. The resulting plasmids were then introducedinto tissue culture cells (29) using a DEAE-dextran technique (61)modified such that the transfection in the presence of DEAE-dextran wascarried out for 8 hours. Cell media was changed every 2-3 days. 200microliters was removed daily for interferon bioassay. Typical yieldswere 50-100 units/ml on samples assayed three or four days aftertransfection.

The product of expression lacks the CYS-TYR-CYS N-terminal portion ofrecombinant human immune interferon (Compare FIG. 5), supporting theoccurrence of signal peptide cleavage at the CYS-GLN junction (aminoacids 3 and 4 in FIG. 5) such that the mature polypeptide would in factconsist of 143 amino acids.

N. Partial Purification of des-CYS-TYR-CYS Recombinant Human interferon

In order to produce greater quantities of the des-CYS-LYS-CYSrecombinant human immune interferon, fresh monolayers of COS-7 cells inten 10 cm plates were transfected with a total of 30 μg pDLIF3 in 110mls DEAE-Dextran (200 μg/ml DEAE Dextran 500,000 MW, 0.05M Tris pH 7.5,in DMEM). After 16 hrs. at 37°, the plates were washed twice with DMEM.15 mls fresh DMEM supplemented with 10 percent f.b.s., 2 mM glutamine,50 μ/ml penicillin G, and 50 mg/ml streptomycin was then added to eachplate. The media was replaced the following day with serum-free DMEM.Fresh serum-free media was then added every day. The media collected waskept at 4° until either assayed or bound to CPG. The pooled fractionsfrom 3 and 4 day post-transfection samples were found to containessentially all of the activity.

0.5 g of CPG (controlled pore glass, Electronucleonics, CPG 350, meshsize 120/200) were added to 100 ml of cell supernatant and the mixturestirred for 3 hrs at 4° C. After a short centrifugation in a bench topcentrifuge the settled beads were packed into a column and thoroughlywashed with 20 mM NaPO₄ 1M NaCL 0.1 percent β-mercaptoethanol pH 7.2.The activity was then eluted with the same buffer containing 30 percentethyleneglycol followed by further elution with the above buffercontaining 50 percent ethyleneglycol. Basically all the activity boundto the CPG. 75 percent of the eluted activity was found in the fractionseluted with 30 percent ethyleneglycol. These fractions were pooled anddiluted with 20 mM NaPO₄ 1M NaCl pH 7.2 to a final concentration of 10percent ethyleneglycol and directly applied to a 10 ml Con A Sepharose(Pharmacia) column. After a thorough wash with 20 mM NaPO₄ 1M NaCl pH7.2 the activity was eluted with 20 mM NaPO₄ 1M NaCl 0.2Mα-methyl-D-mannoside. A substantial amount of the activity (55 percent)did not bind to this lectin. 45 percent of the activity eluted withα-methyl-D-mannoside.

PHARMACEUTICAL COMPOSITIONS

The compounds of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe human immune interferon product hereof is combined in admixture witha pharmaceutically acceptable carrier vehicle. Suitable vehicles andtheir formulation are described in Remington's Pharmaceutical Sciencesby E. W. Martin, which is hereby incorporated by reference. Suchcompositions will contain an effective amount of the interferon proteinhereof together with a suitable amount of vehicle in order to preparepharmaceutically acceptable compositions suitable for effectiveadministration to the host.

A. Parenteral Administration

The human immune interferon hereof may be parenterally administered tosubjects requiring antitumor, or antiviral treatment, and to thoseexhibiting immunosuppressive conditions. Dosage and dose rate mayparallel that currently in use in clinical investigations of other humaninterferons, e.g., about (1-10)×10⁶ units daily, and in the case ofmaterials of purity greater than 1 percent, likely up to, e.g., 50×10⁶units daily. Dosages of IFN-γ could be significantly elevated forgreater effect owing to the essential absence of human proteins otherthan IIN-γ, which proteins in human derived materials may induce certainuntoward effects.

As one example of an appropriate dosage form for essentially homogeneousIFN-γ in parenteral form applicable herein, 3 mg. IFN-γ of specificactivity of, say, 2×10⁸ U/mg may be dissolved in 25 ml. 5N albumin(human)--USP, the solution passed through a bacteriological filter andthe filtered solution aseptically subdivided into 100 vials, eachcontaining 6×10⁶ units pure interferon suitable for parenteraladministration. The vials are preferably stored in the cold (-20° C.)prior to use.

BIOASSAY DATA A. Characterization of Antiviral Activity

For antibody neutralizations, samples were diluted, if necessary, to aconcentration of 500-1000 units/ml with PBS-BSA. Equal volumes of samplewere incubated for 2-12 hrs at 4 degrees with serial dilutions of rabbitantihuman leukocyte, fibroblast, or immune interferon antisera. Theanti-IFN-α and β were obtained from the National Institute of Allergyand Infectious Diseases. The anti-IFN-γ was prepared using authenticIFN-γ (5-20 percent purity) purified from stimulated peripheral bloodlymphocytes. Samples were centrifuged 3 minutes at 12,000×g for 3 minbefore assay. To test pH 2 stability, samples were adjusted to pH 2 byaddition of 1N HCl, incubated for 2-12 hrs at 4°, and neutralized byaddition of 1N NaOH before assay. To test sodium dodecyl sulfate (SDS)sensitivity, samples were incubated with an equal volume of 0.2 percentSDS for 2-12 hrs at 4° before assay.

B. Characterization of IFN-γ Produced by E. coli and COS-7 cells

    ______________________________________                                                 Antiviral Activity (Units/ml)                                                                               COS-7                                                                E. coli  cell/                                                                W3110/   pSVγ69                                      IFN-   IFN-   IFN- pIFN-γtrp48                                                                      Super-                                 Treatment  α                                                                              β γ                                                                            extract  natant                                 ______________________________________                                        Untreated  375    125    250  250        62.5                                 pH 2       375    125    <6   <12      <4                                     0.1 percent SDS                                                                          375    --     <4   <8       --                                     Rabbit anti-IFN-α                                                                  <8     125    250  250      187                                    Rabbit anti-IFN-β                                                                   375    <8     187  250      125                                    Rabbit anti-IFN-γ                                                                  375    125    <4   <8       <4                                     ______________________________________                                    

This table shows the characteristic behavior of IFN-α, β and γ standardsafter various treatments. The interferon activity produced by E. coliW3110/pIFN-γ trp 48 and by COS-7/pSVγ69 is acid-sensitive,SDS-sensitive, and neutralized by immune interferon antiserum. It is notneutralized by antibodies to IFN-α or β. These data confirm that theproducts produced in these systems are immune interferons and that thecDNA insert of plasmid p69 codes for IFN-γ.

The immune interferon protein hereof has been defined by means ofdetermined DNA gene and deductive amino acid sequencing--cf. FIG. 5. Itwill be understood that for this particular interferon, embraced herein,natural allelic variations exist and occur from individual toindividual. These variations may be demonstrated by (an) amino aciddifference(s) in the overall sequence or by deletions, substitutions,insertions, inversions or additions of (an) amino acid(s) in saidsequence. All such allelic variations are included within the scope ofthis invention.

Notwithstanding that reference has been made to particular preferredembodiments, it will be further unerstood that the present invention isnot to be construed as limited to such, rather to the lawful scope ofthe appended claims.

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What is claimed is:
 1. A DNA molecule comprising a recombinant DNAmolecule or a cDNA molecule encoding a polypeptide comprising the aminoacid sequence: ##STR3##
 2. A DNA molecule comprising a recombinant DNAmolecule or a cDNA molecule encoding a polypeptide comprising the aminoacid sequence: ##STR4##
 3. A DNA molecule according to claim 1 or claim2 operably linked with a DNA sequence capable of effecting expression ofthe DNA encoding said polypeptide.
 4. A molecule consisting essentiallyof a DNA molecule encoding the amino acid sequence of recombinant humanimmune interferon.
 5. A molecule consisting essentially of a DNAmolecule encoding the amino acid sequence of des-CYS-TYR-CYS recombinanthuman immune interferon.